SUMMARY. Bacteria utilize intracellular second messenger signaling systems to sense and appropriately respond to changing environmental conditions. Cyclic di-nucleotides are widespread second messengers found throughout living systems. Cyclic di-GMP (c-di-GMP) the focus of this proposal, is synthesized by 80% of bacteria to control phenotypes such as biofilm formation, motility, and virulence gene expression. Although the synthesis of c-di-GMP is only carried out by enzymes containing a GGDEF domain, degradation of this signal can occur by two distinct mechanisms. EAL enzymes can hydrolyze one bond of the c-di-GMP ring to generate linear 5?-pGpG whereas HD-GYP enzymes hydrolyze both bonds to yield GMP. It has long been hypothesized that 5?-pGpG may function as a signaling molecule in its own right as concentrations of 5?-pGpG would be high upon EAL activation in cells that have high intracellular c-di-GMP (i.e. during biofilm dispersal). But such a signaling role has never been identified. The Sundin and Waters laboratories have shown that c-di- GMP is a key signaling molecule in the plant pathogen Erwinia amylovora inducing biofilm formation and repressing transcription of the hrp Type III Secretion System (T3SS). Transitions between these two phenotypes occur during colonization of host plants and are critical for survival of E. amylovora in the different plant tissues. While studying the mechanism by which c-di-GMP inhibits T3SS expression, we have uncovered evidence that 5?-pGpG activates these genes via a direct interaction with the master regulator HrpS. Specifically, 5?-pGpG is a potent inhibitor of c-di-GMP binding to HrpS and engineered mutant strains of E. amylovora that have high intracellular concentration of 5?-pGpG show elevated T3SS transcription. Our central hypothesis is that 5?-pGpG functions as a coactivator of HrpS in E. amylovora to induce T3SS expression. E. amylovora offers an ideal model system to further explore 5?-pGpG signaling because of its simple c-di-GMP signaling network and the powerful advantage that we can study these signaling systems during host infection, which is the actual environment that they evolved. To further characterize 5?pGpG signaling, in Aim 1 we will perform biochemical analyses to assess 5?-pGpG binding to HrpS both with and without c-di-GMP and determine the impact of 5?-pGpG and c-di-GMP on the ability of HrpS to bind to target promoters. We will also initiate studies to identify the key amino acids in HrpS necessary for c-di-GMP and 5?-pGpG binding. In Aim 2, the functional consequences of c-di-GMP and 5?-pGpG will be examined during plant infection using a series of defined mutants that specifically alter the concentrations of each signal. Furthermore, transcriptomic analysis will be used to identify genes that are regulated by HrpS in a 5?-pGpG-dependent manner. The net results of these studies will be identification of the first 5?-pGpG signaling pathway in any organism. As many important bacterial pathogens encode GGDEF and EAL enzymes, but no HD-GYP enzymes, 5?-pGpG may be an unappreciated but widespread signaling molecule.